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Carlos Pedraz-Valdunciel
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P61 - Tumor Biology and Systems Biology - Basic and Translational Science - KRAS (ID 199)
- Event: WCLC 2020
- Type: Posters
- Track: Tumor Biology and Systems Biology - Basic and Translational Science
- Presentations: 1
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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P61.01 - Imipramine Blue (IP) plus MET Tyrosine Kinase Inhibitors (TKI) Suppress Lung Adenocarcinoma (LUAD) KRAS Mutation Tumor Growth (ID 1348)
00:00 - 00:00 | Author(s): Carlos Pedraz-Valdunciel
- Abstract
Introduction
KRAS mutations in LUAD co-occur with TP53 mutations and LKB1 non-synonymous mutations (nsm), portending a poor prognosis. MET amplification has not been considered. Previously, we identified OTSSP167 as a PAK1 kinase inhibitor with significant activity in A549 (KRASG12-C and LKB1nsm). OTSSP167 plus auranofin (PKCι) shows high synergism and inhibits tumor growth in mice in the H23 cell line (KRASG12-C, p53mut and LKB1nsm) (Ito et al, Cell Comm and Signaling 2019). In addition, OTSSP167 has a potent MELK inhibition effect. We hypothesize that MET expression could be upregulated in KRAS-mutant cell lines, based on the fact that the putative signaling pathway, MELK-forkhead box M1 (FOXM1)-MET, could be present in KRAS mutant cells. In the current study, we examined the combination of MET TKIs with imipramine blue (FOXM1 inhibitor).
Methods
Quantitative real time PCR of MET and FOXM1 was performed in 4 KRAS-mutant cell lines (A549, H23, H460 and Calu6). LKB1 mRNA was assessed in 32 advanced KRAS-mutant LUAD patients. Cell proliferation assays were performed in A549 and H23, and in the EBC1 (MET amplified lung cancer cell line). Synergy was defined by a combination index (CI) of < 0.75 by Chou-Talalay. Cell lines were treated with IP and MET TKIs (crizotinib, savolitinib and tepotinib).
Results
MET mRNA was elevated in A549 and H23 (which both carry LKB1nsm) but not in H460 and Calu6. FOXM1 mRNA was overexpressed in H23. Synergy (CI<0.75) was seen with IP plus tepotinib in the A549 and H23 cell lines, but not in H460 and Calu6. Synergy was also noted with IP plus crizotinib, but not with savolitinib. The CI of IP plus MET TKIs in the EBC1 cell line (which is only MET amplified) was >1. The median overall survival for KRAS-mutant LUAD patients with low LKB1 was 1.1 months versus 19.4 for those with high LKB1 (p=<0.005).
Conclusion
The bona fide of MET TKIs (tepotinib) plus IP in KRAS cell lines with LKB1 nsm, encourages the determination of clinical benefit of tepotinib plus IP in KRAS mutant LUAD patients. The liaison of MET and LKB1 nsm should be further investigated. LKB1 mRNA expression, together with MET and FOXM1 mRNA expression, warrants assessment in KRAS LUAD patients.
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P65 - Tumor Biology and Systems Biology - Basic and Translational Science - NC RNA (ID 204)
- Event: WCLC 2020
- Type: Posters
- Track: Tumor Biology and Systems Biology - Basic and Translational Science
- Presentations: 1
- Moderators:
- Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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P65.04 - Tracking circRNAs in Lung Adenocarcinoma Samples as Promising Biomarkers for Cancer Detection using the NanoString nCounter® (ID 3502)
00:00 - 00:00 | Presenting Author(s): Carlos Pedraz-Valdunciel
- Abstract
Introduction
Lung cancer is positioned as the foremost cause of cancer death at global scale. The lack of reliable biomarkers for early detection seems to be the main cause underlying this high mortality rate; therefore, the discovery of new candidates allowing timely diagnosis of the disease is rather imperative.
Circular RNAs (circRNAs) have strongly emerged as valuable tissue-specific biomarkers of different disorders, including lung cancer. The covalently linked 5´-3´ends provide them not only with a distinctive structure but also keeps them exempted from the exonuclease activity making them very stable. They have been described to be highly expressed in extracellular vesicles (EVs) when compared with mRNA; however, despite of the aforementioned properties, their potential as biomarkers has not been fully explored in lung cancer, yet very far to be implemented in the liquid biopsy settings.
Through this study we demonstrate the feasibility of using the NanoString nCounter® Flex platform for the study of circRNAs in different fresh and formalin-fixed paraffin embedded (FFPE) lung cancer material including EVs, hence paving the way for the development of new diagnosis platforms based on this technology and the different circRNA expression patterns.
Methods
Lung cancer cell lines were cultured under standard laboratory conditions until harvested for RNA extraction. EVs were isolated from the cell culture medium by ultracentrifugation.
FFPE tissue samples were retrospectively collected; samples were macro-dissected and total RNA was extracted.
Patient blood was separated into plasma and cellular fractions by centrifugation. Plasma-derived EVs were isolated using the miRCURY Exosome Serum/Plasma Kit.
RNA from EVs was extracted with TRI-Reagent.
For mRNA depleted samples, a treatment with RNase R was performed.
Gene expression analysis was carried out using the NanoString nCounter® platform with a customized panel harboring 85 circRNA related to the biology of the disease.
Results
First runs showed circRNA expression in lung cancer cell lines. A comparative analysis of total RNA versus mRNA-depleted RNA was performed resulting in an overall circRNA enrichment of the latter. An analogous experiment was performed in FFPE PC9 cells; however, circRNA enrichment was not achieved.
Likewise, FFPE lung cancer tissue samples were analyzed; Consequently, circRNA expression has been evident in all samples analyzed so far.
EV-derived RNA (exRNA) from cell lines was also tested in the nCounter®. Counts corresponding to different transcripts were evident in all samples without previous amplification step. Similar results were found in exRNA from one NSCLC patient. More patient samples are currently being collected to validate these results.
Conclusion
Through this project we have demonstrated the feasibility of using nCounter® for the study of circRNAs in different lung cancer materials. RNase R treatment proved to be beneficial for circRNA enrichment in fresh samples but not FFPE samples, probably due to the mechanical and chemical breakage they may experiment during purification, thus becoming susceptible to RNase R degradation.
CircRNAs were also expressed in EVs; however, more patient samples need to be tested. Pre-amplification steps are advisable in the future to explored the sensitivity of the technology before being implemented into the liquid biopsy settings.
